The expression of different classes of bacteriophage T4 genes (early, middle, and late) is transcriptionally regulated. T4 middle promoters, which have a unique consensus sequence ('MotA box') centered at -30, are transcribed by phage-modified host RNA polymerase in the presence of an activator, the T4 MotA protein. The middle promoter PuvsX has the MotA box at -30 as well as other matches at -35, -51, -70, and -87. DNase I footprints indicate that MotA protects PuvsX from positions -25 to -59 on the template strand and from -19 to -58 on the non-template strand, regions that include the MotA boxes at -30, -35, and -51. However, only the MotA box at -30 is required for activation of transcription. MotA also protects from +40 to +57, a region downstream of the transcription start which does not have a recognizable MotA box. This result suggests that MotA may bind to another motif besides the MotA box. MotA binding to PuvsX is specific, but it is also weak. We assayed the stability of MotA, phage-modified polymerase, or both on PuvsX by observing the resistance of the protein-DNA complex to HindIII, which cleaves within the MotA protected region and near sites where unmodified RNA polymerase contacts E. coli promoters. We find that the complex formed in the presence of MotA and polymerase is more stable (half-life of 2.5 min) than that made with polymerase alone (half-life of 1.4 min). Under these conditions, no protection by MotA alone is detected. We speculate that MotA makes unstable contacts at the MotA box at -30 which are then stabilized by its interaction with phage-modified polymerase. Although not encoded by an intron, the T4 SegA protein shares amino acid motifs with a family of fungal and phage proteins found within mobile group I introns. We have purified the SegA protein to homogeneity and find that it has a DNA endonuclease activity that cleaves DNA with a hierarchy of site specificity. PCR analyses indicate that several T-even related phages lack the segA gene, and a segA amber mutant of bacteriophage T4 that we constructed had no detectable phenotype. Taken together, our results support the hypothesis that the segA gene of T4 originated from the insertion of a mobile, endonuclease-encoding DNA into an intergenic region of the T4 genome.